The goal of this project is to develop technologies for transferring large gene clusters (30-300 kb) from a distantly related microorganism to Escherichia coli such that an entire metabolic pathway can be grafted to the host. This technology will be demonstrated by moving genes specifying two pathways, anaerobic benzoate degradation and nitrogen fixation, from Rhodopseudomonas palustris to E. coli. The firstpathway converts benzoate to pimeloyl-coA, which is the essential precursor for biotin synthesis in E. coli. The resulting strain provides a novel path for biosynthesis of biotin from aromatic compounds. The second pathway allows E. coli to fix molecular nitrogen, a new life style for this host, and paves the way for future nitrogenase-catalyzed hydrogen production. Intellectual Merit: This technology enables the exploration of a vast repository of metabolic capability in poorly characterized, but sequenced bacteria. By moving the entire pathway to a well-known and fast- growing host, the metabolic capability can be readily studied and utilized in an industrial scale. Typically, large gene clusters from a distantly related organism are not expressedefficiently in E. coli for several reasons, including the large size of the foreign DNAfragment posing difficulty in cloning, as well as the incompatibility and inability of promoter and control sequences to be recognized by E. coli RNA polymerase and transcription factors. Our strategy here is inspired by two natural processes: horizontal gene transfer during bacterial evolution and phage infection. To achieve horizontal gene transfer, an efficient method for transferring large gene clusters will be developed based on a yeast recombination system and bacterial artificial chromosome (BAG). In addition, we will identify and clone the essential transcription elements from the donor organism into E. coli such that the new host can readily express the donor gene cluster without individual optimization (which resembles the strategy used by bacterial phages during infection). One of the resulting strains will be able to convert benzoate to biotin in E. coli, providing a novel route to by-pass the bottleneck of precursor supply in biotin production. The other resulting strain will be able to utilize N2 as the sole nitrogen source, demonstrating a new life-style for E. coli and establishing the potential for further metabolic engineering for hydrogen production. Broader Impact: The proposed research provides a new way for studying metabolic functions in uncharacterized microorganisms and a basis for training and educating new generations of scientists and engineers. In particular, selected projects proposed here will be used as modules in a workshop established by FRT at Ohio State and form the basis for a course (Metabolic Engineering) at UCLA.